The mu-opioid receptor (MOR) mediates most of the actions of morphine and other clinically relevant analgesics as well as drugs of abuse such as heroin. The proteomic and functional studies proposed in this application are designed to elucidate the protein components of MOR signaling complexes. Identification of MOR interacting proteins will allow us to test our hypothesis that protein-protein interactions play an important role in regulating the MOR signal transduction pathway. In Aim I, we will identify and characterize MOR interacting proteins. A split-ubiquitin screen we have performed identified several novel MORIPs including GPR177, the mammalian ortholog of Drosophila Wntless. To identify additional MORIPs, each of the intracellular domains of the human MOR will be used as bait to separately screen a human brain cDNA library. We also propose to utilize highly selective anti-MOR antibodies combined with proteomics and mass spectrometry to identify a spectrum of MORIPs from immunoprecipitated mouse brain lysates. The major goal in Aim 2 will be to validate the MOR-protein interactions identified in Aim I. Cellular colocalization studies will confirm whether or not the MOR and candidate interactors are expressed within the same cells and intracellular compartments. Pull-down and coimmunoprecipitation will substantiate the MOR-protein interaction, while deletion mapping will permit identification of sites within the proteins that are necessary for the interactions to occur. Preliminary studies we have so far performed indicate that GPR177 meets all inclusion criteria and appears to represent a bona-fide MORIP. The goal in Aim 3 is to understand the functional significance of MOR/GPR177 interaction. We will examine the requirement for MOR/GPR177 interaction in MOR trafficking, desensitization, and signal transduction. We will also examine the role of the MOR/GPR177 interaction in regulating Wnt2 secretion from cells. To accomplish this goal, we will determine whether disrupting the MOR/GPR177 interaction, by expressing dominant negative forms of GPR177 or by knocking down GPR177 expression, affects the functional properties of the MOR. Identification of MOR interacting proteins will provide new insights into the etiology of drug abuse and dependence. RELEVANCE (See instructions): This project seeks to identify and evaluate the function of proteins that interact with and regulate the muopioid receptor (MOR), the cell associated receptor that mediates most of the analgesic actions of morphine as well as drugs of abuse. Understanding the function of GPR177, a novel interacting protein we identified, is likely to provide new insight into the etiology of drug abuse and dependence. MOR-interacting proteins may also represent novel therapeutic targets for the treatment of these important public health problems.